Analysis of drug/plasma protein interactions by means of asymmetrical flow field-flow fractionation.
OBJECTIVE
The applicability of Asymmetrical Flow Field-Flow Fractionation (Asymmetrical Flow FFF) as an alternative tool to examine the distribution of a lipophilic drug (N-Benzoyl-staurosporine) within human plasma protein fractions was investigated with respect to high separation speed and loss of material on surfaces due to adsorption.


METHODS
Field-Flow Fractionation is defined as a group of pseudochromatographic separation methods, where compounds are separated under the influence of an externally applied force based on differences in their physicochemical properties. This method was used to separate human plasma in its protein fractions. The drug distribution in the fractions was investigated by monitoring the fractionated eluate for drug content by fluorescence spectroscopy.


RESULTS
Human plasma was separated into human serum albumin (HSA), high density lipoprotein (HDL), alpha 2-macroglobulin and low density lipoprotein (LDL) fractions in less than ten minutes. Calibration of the system and identification of the individual fractions was performed using commercially available protein reference standards. The influence of membrane type and carrier solution composition on the absolute recovery of N-Benzoyl-staurosporine and fluorescein-isothiocyanate-albumin (FITC-albumin) was found to be quite significant. Both factors were optimized during the course of the investigations. N-Benzoyl-staurosporine was found to be enriched in the fraction containing HSA.


CONCLUSIONS
If experimental conditions are thoroughly selected and controlled to suppress drug and plasma protein adsorption at the separation membrane, Asymmetrical Flow FFF shows high recoveries and fast separation of human plasma proteins, and can be a reliable tool to characterize drug/plasma protein interactions. For analytical purposes it has the potential to rival established technologies like ultracentrifugation in terms of ease-of-use, precision, and separation time.
Madörin
M
M
Novartis Pharma AG, Basel, Switzerland.
van Hoogevest
P
P
Hilfiker
R
R
Langwost
B
B
Kresbach
G M
GM
Ehrat
M
M
Leuenberger
H
H
D000327:Adsorption; D005591:Chemical Fractionation / Q000379:methods; D002845:Chromatography; D004791:Enzyme Inhibitors / Q000737:chemistry*; D006801:Humans; D008075:Lipoproteins, HDL / Q000302:isolation & purification*; D008077:Lipoproteins, LDL / Q000302:isolation & purification*; D008567:Membranes, Artificial; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012015:Reference Standards; D012709:Serum Albumin / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000737:chemistry; D000511:alpha-Macroglobulins / Q000302:isolation & purification*
D004791:Enzyme Inhibitors; D008075:Lipoproteins, HDL; D008077:Lipoproteins, LDL; D008567:Membranes, Artificial; D012709:Serum Albumin; D000511:alpha-Macroglobulins; D011493:Protein Kinase C; D019311:Staurosporine; C059539:midostaurin